Chemokines are small, secreted pro-inflammatory proteins, which mediate directional migration of leukocytes from the blood to the site of injury. Depending on the position of the conserved cysteines characterizing this family of proteins, the chemokine family can be divided structurally in C, C—C, C—X—C and C—X3—C chemokines which bind to a series of membrane receptors (Baggiolini M et al., 1997; Fernandez E J and Lolis E, 2002).
These membrane receptors, all heptahelical G-protein coupled receptors, allow chemokines to exert their biological activity on the target cells, which may present specific combinations of receptors according to their state and/or type. The physiological effects of chemokines result from a complex and integrated system of concurrent interactions: the receptors often have overlapping ligand specificity, so that a single receptor can bind different chemokines, as well a single chemokine can bind to different receptors.
Usually chemokines are produced at the site of injury and cause leukocyte migration and activation, playing a fundamental role in inflammatory, immune, homeostatic, hematopoietic, and angiogenic processes. Even though there are potential drawbacks in using chemokines as therapeutic agents (tendency to aggregate and promiscuous binding, in particular), these molecules are considered good target candidates for therapeutic intervention in diseases associated to such processes, by inhibiting specific chemokines and their receptors at the scope to preventing the excessive recruitment and activation of leukocytes (Baggiolini M, 2001; Loetscher P and Clark-Lewis I, 2001; Godessart N and Kunkel S L, 2001).
Studies on structure-activity relationships indicate that chemokines have two main sites of interaction with their receptors, the flexible amino-terminal region and the conformationally rigid loop that follows the second cysteine. Chemokines are thought to dock onto receptors by means of the loop region, and this contact is believed to facilitate the binding of the amino-terminal region that results in receptor activation. This importance of the amino-terminal region has been also demonstrated by testing natural and synthetic chemokines in which this domain is modified or shortened. This processing, following proteolytic digestion, mutagenesis, or chemical modification of amino acids, can either activate or render these molecules inactive, generating compounds with agonistic and/or antagonistic activity. Thus, chemokines with specific modifications in the amino-terminal region have therapeutic potential for inflammatory and autoimmune diseases (Schwarz and Wells, 1999).
CCL2, also known as Monocyte Chemoattractant Protein 1 (MCP-1) or Monocyte Chemotactic And Activating Factor (MCAF), has been identified as having a central role in inflammation, being capable of promoting the recruitment of monocytes and lymphocytes in response to injury and infection signals in various inflammatory diseases, different types of tumors, cardiac allograft, AIDS, and tuberculosis (Yoshimura T et al., 1989; Gu L et al., 1999). The physiological activities associated with CCL2 have been extensively studied by means of transgenic animals, which allowed the demonstration that this chemokine controls not only monocyte recruitment in inflammatory models, but also the expression of cytokines relate d to T helper responses and the initiation of atherosclerosis (Gu L et al., 2000; Gosling J et al, 1999; Lu B et al, 1998).
Structurally, CCL2 consists of a N-terminal loop and a beta sheet overlaid by an alpha helix at the C-terminal end, and forms homodimers, even though has been detected as a monomer in specific experimental conditions (Handel T et al., 1996; Kim K S et al., 1996; Lubkowski J, et al., 1997). As for many other chemokines, the literature provides many examples of structure-activity studies in which CCL2 mutants have been generated by expressing N-terminal truncated or single site substituted variants to assess the role of the deleted or substituted amino acids in CCL2-associated binding activities and other properties (Gong J and Clark-Lewis I, 1995; Zhang Y et al., 1996; Steitz S A et al., 1998; Gu L et al., 1999; Hemmerich S et al., 1999; Seet B T et al., 2001).
In particular, the role of dimerization in CCL2 receptor binding and activation was studied showing that different mutations in the terminal region hindering dimerization may alter some CCL2 activities such as receptor binding affinity, stimulation of chemotaxis, inhibition of adenylate cyclase, and stimulation of calcium influx (Paavola C et al, 1998). While one mutant described by Paavola, herein called P8A*, does not dimerize, but maintains original potency and efficacy, another obligate monomeric mutant described by Paavola, herein called Y13A*, was shown to have a 100-fold weaker binding affinity in vitro, to be a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx in vitro, and unable to stimulate chemotaxis in cell culture. Similarly to Y13A*, a mutant, [1+9-76]MCP-1 (a CCL2 variant lacking residues 2-8), antagonizes CCL2 activities in vitro.
The binding properties of the CCL2 mutant containing the P8A substitution were also studied in an experimental model based upon the recognition of the viral chemokine binding protein M3, demonstrating the efficient binding of this viral protein to this CCL2 mutant (Alexander J M et al., 2002). Moreover it has been shown that monomeric variants of chemokines, such as CCL2-P8A, are devoid of activity in vivo, although fully active and indistinguishable from the dimeric form in vitro (Proudfoot A et al., 2003).
Examples of CCL2 mutants involving residues not affecting the dimerization profile of the resulting protein have been already described in the literature as leading to molecules having antagonistic properties towards CCL2 (U.S. Pat. No. 5,739,103, WO 03/84993). However, there is not indication in the prior art that a specific chemokine mutant, and in particular a CCL2 mutant, being an obligate monomer due to a single site substitution (for example, involving a Proline), may act as a chemokine antagonist.